Rotaviruses have a double stranded segmented RNA genome and hence undergo genetic reassortment at high frequency. We have produced a series of temperature sensitive mutants of both bovine (UK) and rhesus rotavirus. We have used such ts mutants to isolate reassortant rotaviruses from the growth yield of cells coinfected with a variety of human rotaviruses and ts bovine rotavirus. These reassortants were used to ascertain the gene coding assignments for human rotavirus neutralization specificity and growth restriction in tissue culture. Also, the human-bovine rotavirus reassortants were used to define the serotypic diversity of human rotaviruses. We isolated a series of reassortants derived from cells coinfected with ts bovine and ts rhesus rotaviruses. Characterization of these reassortants showed that: (1) neutralization specificity and hemagglutination reassort independently and are coded for by different genes (8 or 9 and 4 respectively); (2) protease enhancement of virus replication co-segregates with the viral hemagglutintin and is coded for by the same gene, i.e., gene 4. Finally, using highly specific hyperimmune or monoclonal antibodies, we have isolated a series of reassortants derived from a cross of wild type bovine (UK) or RRV with human rotavirus (serotype 1, 2, 3 or 4). These reassortants that were isolated in primary tissue, possess only one or two human rotavirus genes. One of these genes codes for the glycoprotein that induces neutralizing antibody and thus these reassortants represent prime candidates for use in a live attenuated vaccine.